Primary transcripts in eukaryotes often contain intervening sequences, which must be excised to generate functional messenger RNAs. Nuclear pre-mRNA splicing is thus an essential step in regulating gene expression. Alternative splicing events play a role in generating a broad spectrum of genetic diversity in higher eukaryotes and determine normal cell development. Disruption of splicing patterns is often associated with disease. Although much progress has been made in defining the general features of splicing as well as identifying specific components, understanding the regulation of this process will require the analysis of the splicing machinery on the molecular level. Elucidation of RNA-RNA and RNA-protein interactions and the way in which conformational changes are achieved in the spliceosome is central to this understanding. Insights into these questions can be gained by studying the molecular interactions of ATPases, such as Prp16, Prp22 and Prp43 known to function at specific steps of splicing. This proposal presents experiments to delineate the mechanisms by which spliceosomal DEAH-box proteins use ATP hydrolysis to promote the final step of the splicing reaction leading to the formation of mature RNA and of the ensuing steps to release the products of the reaction from the spliceosome. The principal investigator proposes to dissect the splicing pathway by reconstituting the partial reactions from purified components. The choice of yeast as the experimental system permits the powerful combination of genetic and biochemical approaches in studying the molecular interactions in complex processes. Given the large degree of evolutionary conservation between yeast and mammals in the structure and function of the basic splicing apparatus, the proposed studies will be broadly relevant to pre-mRNA splicing in higher eukaryotes. Moreover, DExH/D-box proteins play important roles in all major nucleic acid transactions, and the proposed analysis of the spliceosomal DExH-box proteins will likely give insight into the mechanisms and functions of other family members.